33 results on '"Andrew R. J. Lawson"'
Search Results
2. Development of a highly sensitive liquid biopsy platform to detect clinically-relevant cancer mutations at low allele fractions in cell-free DNA.
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Davina Gale, Andrew R J Lawson, Karen Howarth, Mikidache Madi, Bradley Durham, Sarah Smalley, John Calaway, Shannon Blais, Greg Jones, James Clark, Peter Dimitrov, Michelle Pugh, Samuel Woodhouse, Michael Epstein, Ana Fernandez-Gonzalez, Alexandra S Whale, Jim F Huggett, Carole A Foy, Gerwyn M Jones, Hadas Raveh-Amit, Karin Schmitt, Alison Devonshire, Emma Green, Tim Forshew, Vincent Plagnol, and Nitzan Rosenfeld
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Medicine ,Science - Abstract
Detection and monitoring of circulating tumor DNA (ctDNA) is rapidly becoming a diagnostic, prognostic and predictive tool in cancer patient care. A growing number of gene targets have been identified as diagnostic or actionable, requiring the development of reliable technology that provides analysis of multiple genes in parallel. We have developed the InVision™ liquid biopsy platform which utilizes enhanced TAm-Seq™ (eTAm-Seq™) technology, an amplicon-based next generation sequencing method for the identification of clinically-relevant somatic alterations at low frequency in ctDNA across a panel of 35 cancer-related genes.We present analytical validation of the eTAm-Seq technology across two laboratories to determine the reproducibility of mutation identification. We assess the quantitative performance of eTAm-Seq technology for analysis of single nucleotide variants in clinically-relevant genes as compared to digital PCR (dPCR), using both established DNA standards and novel full-process control material.The assay detected mutant alleles down to 0.02% AF, with high per-base specificity of 99.9997%. Across two laboratories, analysis of samples with optimal amount of DNA detected 94% mutations at 0.25%-0.33% allele fraction (AF), with 90% of mutations detected for samples with lower amounts of input DNA.These studies demonstrate that eTAm-Seq technology is a robust and reproducible technology for the identification and quantification of somatic mutations in circulating tumor DNA, and support its use in clinical applications for precision medicine.
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- 2018
- Full Text
- View/download PDF
3. Increased somatic mutation burdens in normal human cells due to defective DNA polymerases
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Sigurgeir Olafsson, Inigo Martincorena, Bernard C H Lee, Sara Galavotti, Philip S. Robinson, Federico Abascal, Lynn Martin, Michael R. Stratton, Claire Palles, Raheleh Rahbari, James Hewinson, Andrew R. J. Lawson, Luiza Moore, Mathijs A. Sanders, Ian Tomlinson, Emily Mitchell, Tim H. H. Coorens, Peter J. Campbell, Henry Lee-Six, Claudia Maria Assunta Pinna, Robinson, Philip S. [0000-0002-6237-7159], Coorens, Tim H. H. [0000-0002-5826-3554], Abascal, Federico [0000-0002-6201-1587], Lee-Six, Henry [0000-0003-4831-8088], Moore, Luiza [0000-0001-5315-516X], Pinna, Claudia M. A. [0000-0002-5618-7842], Rahbari, Raheleh [0000-0002-1839-7785], Campbell, Peter J. [0000-0002-3921-0510], Martincorena, Iñigo [0000-0003-1122-4416], Tomlinson, Ian [0000-0003-3037-1470], Stratton, Michael R. [0000-0001-6035-153X], Apollo - University of Cambridge Repository, Hematology, Robinson, Philip S [0000-0002-6237-7159], Coorens, Tim HH [0000-0002-5826-3554], Pinna, Claudia MA [0000-0002-5618-7842], Campbell, Peter J [0000-0002-3921-0510], and Stratton, Michael R [0000-0001-6035-153X]
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Premature aging ,Adult ,Adolescent ,DNA polymerase ,Somatic cell ,Embryonic Development ,medicine.disease_cause ,Germline ,Article ,Gastrointestinal cancer ,Young Adult ,Germline mutation ,SDG 3 - Good Health and Well-being ,Intestinal Neoplasms ,Genetics ,medicine ,Humans ,Germ-Line Mutation ,Phylogeny ,Aged ,DNA Polymerase III ,Mutation ,POLD1 ,biology ,Genome, Human ,Stem Cells ,631/443/7 ,Genomics ,DNA Polymerase II ,Mutation Accumulation ,Middle Aged ,631/67/1504 ,Intestines ,Ageing ,Mutagenesis ,631/208/212 ,biology.protein - Abstract
Funder: Wellcome Clinical PhD fellowship, Funder: Wellcome PhD Studentship, Funder: Jean Shank/Pathological Society Intermediate Fellowship, Mutation accumulation in somatic cells contributes to cancer development and is proposed as a cause of aging. DNA polymerases Pol ε and Pol δ replicate DNA during cell division. However, in some cancers, defective proofreading due to acquired POLE/POLD1 exonuclease domain mutations causes markedly elevated somatic mutation burdens with distinctive mutational signatures. Germline POLE/POLD1 mutations cause familial cancer predisposition. Here, we sequenced normal tissue and tumor DNA from individuals with germline POLE/POLD1 mutations. Increased mutation burdens with characteristic mutational signatures were found in normal adult somatic cell types, during early embryogenesis and in sperm. Thus human physiology can tolerate ubiquitously elevated mutation burdens. Except for increased cancer risk, individuals with germline POLE/POLD1 mutations do not exhibit overt features of premature aging. These results do not support a model in which all features of aging are attributable to widespread cell malfunction directly resulting from somatic mutation burdens accrued during life.
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- 2021
4. Extensive phylogenies of human development inferred from somatic mutations
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Michael R. Stratton, Raheleh Rahbari, Ayesha Noorani, Tim H. H. Coorens, Yvette Hooks, Philip S. Robinson, Thomas R. W. Oliver, Thomas J. Mitchell, Moritz J. Przybilla, Matthew D. C. Neville, Peter J. Campbell, James Hewinson, Rebecca C. Fitzgerald, Inigo Martincorena, Andrew R. J. Lawson, Joseph Christopher, Alex Cagan, Luiza Moore, Rashesh Sanghvi, and Michael Spencer Chapman
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Multidisciplinary ,Zygote ,Lineage (genetic) ,Cell division ,Somatic cell ,Evolutionary biology ,Progenitor cell ,Biology ,Embryonic stem cell ,Germline ,Progenitor - Abstract
Starting from the zygote, all cells in the human body continuously acquire mutations. Mutations shared between different cells imply a common progenitor and are thus naturally occurring markers for lineage tracing1,2. Here we reconstruct extensive phylogenies of normal tissues from three adult individuals using whole-genome sequencing of 511 laser capture microdissections. Reconstructed embryonic progenitors in the same generation of a phylogeny often contribute to different extents to the adult body. The degree of this asymmetry varies between individuals, with ratios between the two reconstructed daughter cells of the zygote ranging from 60:40 to 93:7. Asymmetries pervade subsequent generations and can differ between tissues in the same individual. The phylogenies resolve the spatial embryonic patterning of tissues, revealing contiguous patches of, on average, 301 crypts in the adult colonic epithelium derived from a most recent embryonic cell and also a spatial effect in brain development. Using data from ten additional men, we investigated the developmental split between soma and germline, with results suggesting an extraembryonic contribution to primordial germ cells. This research demonstrates that, despite reaching the same ultimate tissue patterns, early bottlenecks and lineage commitments lead to substantial variation in embryonic patterns both within and between individuals. Somatic mutations obtained from laser microdissected biopsies of human tissues are used to reconstruct the developmental phylogenies of these tissues back to the zygote.
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- 2021
5. Reliable detection of somatic mutations in solid tissues by laser-capture microdissection and low-input DNA sequencing
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Ben Farr, Robert J. Osborne, Mathijs A. Sanders, Alex Cagan, Michael R. Stratton, Inigo Martincorena, Peter J. Campbell, Peter R. Ellis, Tim Butler, Andrew R. J. Lawson, Henry Lee-Six, Tim H. H. Coorens, Simon F. Brunner, Luiza Moore, and Hematology
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0303 health sciences ,Mutation ,Computer science ,Sequence analysis ,Tissue Processing ,Computational biology ,medicine.disease_cause ,Genome ,General Biochemistry, Genetics and Molecular Biology ,DNA sequencing ,03 medical and health sciences ,0302 clinical medicine ,SDG 3 - Good Health and Well-being ,medicine ,Genomic library ,030217 neurology & neurosurgery ,Microdissection ,030304 developmental biology ,Laser capture microdissection - Abstract
Somatic mutations accumulate in healthy tissues as we age, giving rise to cancer and potentially contributing to ageing. To study somatic mutations in non-neoplastic tissues, we developed a series of protocols to sequence the genomes of small populations of cells isolated from histological sections. Here, we describe a complete workflow that combines laser-capture microdissection (LCM) with low-input genome sequencing, while circumventing the use of whole-genome amplification (WGA). The protocol is subdivided broadly into four steps: tissue processing, LCM, low-input library generation and mutation calling and filtering. The tissue processing and LCM steps are provided as general guidelines that might require tailoring based on the specific requirements of the study at hand. Our protocol for low-input library generation uses enzymatic rather than acoustic fragmentation to generate WGA-free whole-genome libraries. Finally, the mutation calling and filtering strategy has been adapted from previously published protocols to account for artifacts introduced via library creation. To date, we have used this workflow to perform targeted and whole-genome sequencing of small populations of cells (typically 100–1,000 cells) in thousands of microbiopsies from a wide range of human tissues. The low-input DNA protocol is designed to be compatible with liquid handling platforms and make use of equipment and expertise standard to any core sequencing facility. However, obtaining low-input DNA material via LCM requires specialized equipment and expertise. The entire protocol from tissue reception through whole-genome library generation can be accomplished in as little as 1 week, although 2–3 weeks would be a more typical turnaround time.
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- 2021
6. Somatic mutation rates scale with lifespan across mammals
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Alex Cagan, Adrian Baez-Ortega, Natalia Brzozowska, Federico Abascal, Tim H. H. Coorens, Mathijs A. Sanders, Andrew R. J. Lawson, Luke M. R. Harvey, Shriram Bhosle, David Jones, Raul E. Alcantara, Timothy M. Butler, Yvette Hooks, Kirsty Roberts, Elizabeth Anderson, Sharna Lunn, Edmund Flach, Simon Spiro, Inez Januszczak, Ethan Wrigglesworth, Hannah Jenkins, Tilly Dallas, Nic Masters, Matthew W. Perkins, Robert Deaville, Megan Druce, Ruzhica Bogeska, Michael D. Milsom, Björn Neumann, Frank Gorman, Fernando Constantino-Casas, Laura Peachey, Diana Bochynska, Ewan St. John Smith, Moritz Gerstung, Peter J. Campbell, Elizabeth P. Murchison, Michael R. Stratton, Iñigo Martincorena, Cagan, Alex [0000-0002-7857-4771], Abascal, Federico [0000-0002-6201-1587], Coorens, Tim H. H. [0000-0002-5826-3554], Lawson, Andrew R. J. [0000-0003-3592-1005], Bhosle, Shriram [0000-0002-7638-2899], Jones, David [0000-0002-0407-0386], Butler, Timothy M. [0000-0001-5803-1035], Spiro, Simon [0000-0002-9621-2192], Milsom, Michael D. [0000-0002-3567-254X], Bochynska, Diana [0000-0003-0219-8818], Smith, Ewan St. John [0000-0002-2699-1979], Gerstung, Moritz [0000-0001-6709-963X], Campbell, Peter J. [0000-0002-3921-0510], Murchison, Elizabeth P. [0000-0001-7462-8907], Stratton, Michael R. [0000-0001-6035-153X], Martincorena, Iñigo [0000-0003-1122-4416], Apollo - University of Cambridge Repository, Hematology, Coorens, Tim HH [0000-0002-5826-3554], Lawson, Andrew RJ [0000-0003-3592-1005], Butler, Timothy M [0000-0001-5803-1035], Milsom, Michael D [0000-0002-3567-254X], Smith, Ewan St John [0000-0002-2699-1979], Campbell, Peter J [0000-0002-3921-0510], Murchison, Elizabeth P [0000-0001-7462-8907], Stratton, Michael R [0000-0001-6035-153X], Smith, Ewan [0000-0002-2699-1979], and Murchison, Elizabeth [0000-0001-7462-8907]
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Scale (anatomy) ,Somatic cell ,Longevity ,Normal tissue ,45/23 ,631/67/69 ,Biology ,13 ,14 ,Oxidative damage ,Germline mutation ,Mutation Rate ,SDG 3 - Good Health and Well-being ,Animals ,Humans ,631/208/212/748 ,Mammals ,Multidisciplinary ,45 ,Mutagenesis ,631/443/7 ,article ,45/77 ,Ageing ,Evolutionary biology ,Mutation ,631/208/212 ,14/63 ,Trait - Abstract
The rates and patterns of somatic mutation in normal tissues are largely unknown outside of humans1–7. Comparative analyses can shed light on the diversity of mutagenesis across species, and on long-standing hypotheses about the evolution of somatic mutation rates and their role in cancer and ageing. Here we performed whole-genome sequencing of 208 intestinal crypts from 56 individuals to study the landscape of somatic mutation across 16 mammalian species. We found that somatic mutagenesis was dominated by seemingly endogenous mutational processes in all species, including 5-methylcytosine deamination and oxidative damage. With some differences, mutational signatures in other species resembled those described in humans8, although the relative contribution of each signature varied across species. Notably, the somatic mutation rate per year varied greatly across species and exhibited a strong inverse relationship with species lifespan, with no other life-history trait studied showing a comparable association. Despite widely different life histories among the species we examined—including variation of around 30-fold in lifespan and around 40,000-fold in body mass—the somatic mutation burden at the end of lifespan varied only by a factor of around 3. These data unveil common mutational processes across mammals, and suggest that somatic mutation rates are evolutionarily constrained and may be a contributing factor in ageing.
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- 2022
7. Extensive heterogeneity in somatic mutation and selection in the human bladder
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Alexandra Colquhoun, Andrew Menzies, Andrew R. J. Lawson, Jose M. C. Tubio, Michael R. Stratton, Luiza Moore, Tim H. H. Coorens, Krishnaa T. Mahbubani, Inigo Martincorena, William Turner, Nicholas Williams, Laura O’Neill, Luke M. R. Harvey, Peter J. Campbell, Jyoti Nangalia, Kourosh Saeb-Parsy, Harald Vöhringer, Bethany Bareham, Sonia Zumalave, Calli Latimer, Vincent Gnanapragasam, Benjamin Thomas, Federico Abascal, Alex Cagan, Mathijs A. Sanders, Anne Y. Warren, Tim Butler, Keiran Raine, Doris Rassl, Yvette Hooks, Moritz Gerstung, Thomas J. Mitchell, and Hematology
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Adult ,Male ,APOBEC ,Biopsy ,Urinary Bladder ,Mutagenesis (molecular biology technique) ,medicine.disease_cause ,Germline mutation ,medicine ,Humans ,APOBEC Deaminases ,Selection, Genetic ,Urothelium ,Gene ,Aged ,Genetics ,Mutation ,Multidisciplinary ,Bladder cancer ,Urinary bladder ,Middle Aged ,Chromatin Assembly and Disassembly ,medicine.disease ,medicine.anatomical_structure ,Urinary Bladder Neoplasms ,Mutagenesis ,Female ,Genes, Neoplasm ,Mutagens - Abstract
Genetic profiles of the bladder Depending on the environment of the individual, the human bladder can be exposed to carcinogens as they are flushed through the body. Lawson et al. and Li et al. examined the genetic composition of laser-dissected microbiopsies from normal and cancer cells collected from the urothelium, a specialized epithelium lining the lower urinary tract (see the Perspective by Rozen). These complementary studies identified the mutational landscape of bladder urothelium through various sequencing strategies and identified high mutational heterogeneity within and between individuals and tumors. Both studies identified mutational profiles related to specific carcinogens such as aristolochic acid and the molecules found in tobacco. These studies present a comprehensive description of the diverse mutational landscape of the human bladder in health and disease, unraveling positive selection for cancer-causing mutations, a diversity of mutational processes, and large differences across individuals. Science , this issue p. 75 , p. 82 ; see also p. 34
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- 2020
8. Multi-regional characterisation of renal cell carcinoma and microenvironment at single cell resolution
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Joana B. Neves, Anne Y. Warren, Tim Butler, Georgina Bowyer, Grant D. Stewart, Andrew R. J. Lawson, Yvette Hooks, Kevin W. Loudon, Matthew D. Young, Liam Bolt, Thomas J. Mitchell, Tev Aho, Lira Mamanova, Antony C. P. Riddick, Eirini S. Fasouli, Sarah J. Welsh, Sarah A. Teichmann, Vincent Gnanapragasam, Maxine G. B. Tran, Ruoyan Li, Sam Behjati, Menna R. Clatworthy, John R. Ferdinand, James N. Armitage, Peter J. Campbell, Thomas R. W. Oliver, and Kerstin B. Meyer
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Transcriptome ,medicine.anatomical_structure ,Somatic cell ,Renal cell carcinoma ,T cell ,Cancer cell ,Cell ,medicine ,Cancer research ,Biology ,medicine.disease ,CD8 ,Exome sequencing - Abstract
Tumour behaviour is dependent on the oncogenic properties of cancer cells and their multi-cellular interactions. These dependencies were examined through 270,000 single cell transcriptomes and 100 micro-dissected whole exomes obtained from 12 patients with kidney tumours. Tissue was sampled from multiple regions of tumour core, tumour-normal interface, normal surrounding tissues, and peripheral blood. We found the principal spatial location of CD8+ T cell clonotypes largely defined exhaustion state, with clonotypic heterogeneity not explained by somatic intra-tumoural heterogeneity. De novo mutation calling from single cell RNA sequencing data allows us to lineage-trace and infer clonality of cells. We discovered six meta-programmes that distinguish tumour cell function. An epithelial-mesenchymal transition meta-programme, enriched at the tumour-normal interface appears modulated through macrophage expressed IL1B, potentially forming a therapeutic target.
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- 2021
9. Author response: Patterns of within-host genetic diversity in SARS-CoV-2
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Naomi Park, Ian Goodfellow, Iliana Georgana, Gerry Tonkin-Hill, Fahad A Khokhar, Luke W. Meredith, Michael Spencer Chapman, Andrew R. J. Lawson, Moritz Gerstung, Rachel Nelson, Cordelia Langford, Grant Hall, Sónia Gonçalves, Alex Alderton, Sarah L Caddy, Laura G Caller, Michael A. Quail, Yasmin Chaudhry, Ian Johnston, Jeffrey C. Barrett, David K. Jackson, Martin D. Curran, Surendra Parmar, M. Estée Török, Dominic P. Kwiatkowski, Stephen D. Bentley, Roberto Amato, Myra Hosmillo, William L Hamilton, Theresa Feltwell, John Sillitoe, Ewan M. Harrison, Aminu S Jahun, Stefanie V Lensing, Anna Yakovleva, Inigo Martincorena, Charlotte J. Houldcroft, Malte L Pinckert, and Cristina V. Ariani
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Genetics ,Genetic diversity ,Host (biology) ,Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ,Biology - Published
- 2021
10. Somatic mutation landscapes at single-molecule resolution
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Michael R. Stratton, Krishnaa T. Mahubani, Raheleh Rahbari, Sigurgeir Olafsson, Kourosh Saeb-Parsy, Emily G. Mitchell, Eugene Jing Kwa, Alex Cagan, David G. Kent, Heather E. Machado, Robert J. Osborne, David T. Jones, Peter J. Campbell, Andrew Russell, Michael Spencer Chapman, Elisa Laurenti, Tim H. H. Coorens, Megan Davies, Yichen Wang, Steven Leonard, Henry Lee-Six, Nina F. Øbro, Adrian Baez-Ortega, Peter R. Ellis, Luke M. R. Harvey, Kieren Allinson, Stefanie V Lensing, Andrew R. J. Lawson, Moritz Gerstung, Federico Abascal, Raul E. Alcantara, Inigo Martincorena, Abascal, Federico [0000-0002-6201-1587], Lawson, Andrew RJ [0000-0003-3592-1005], Russell, Andrew JC [0000-0001-5411-2807], Wang, Yichen [0000-0001-5122-7545], Lee-Six, Henry [0000-0003-4831-8088], Cagan, Alex [0000-0002-7857-4771], Coorens, Tim HH [0000-0002-5826-3554], Jones, David [0000-0002-0407-0386], Mahubani, Krishnaa T [0000-0002-1327-2334], Gerstung, Moritz [0000-0001-6709-963X], Saeb-Parsy, Kourosh [0000-0002-0633-3696], Laurenti, Elisa [0000-0002-9917-9092], Stratton, Michael R [0000-0001-6035-153X], Rahbari, Raheleh [0000-0002-1839-7785], Campbell, Peter J [0000-0002-3921-0510], Osborne, Robert J [0000-0002-1914-1239], Martincorena, Iñigo [0000-0003-1122-4416], and Apollo - University of Cambridge Repository
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Male ,Mutation rate ,Cell division ,Somatic cell ,Colon ,Cellular differentiation ,DNA Mutational Analysis ,Mutagenesis (molecular biology technique) ,Biology ,medicine.disease_cause ,Epithelium ,Cohort Studies ,03 medical and health sciences ,0302 clinical medicine ,Germline mutation ,Mutation Rate ,Alzheimer Disease ,medicine ,Humans ,030304 developmental biology ,Genetics ,Neurons ,0303 health sciences ,Mutation ,Multidisciplinary ,Blood Cells ,Stem Cells ,Cell Differentiation ,Muscle, Smooth ,Middle Aged ,Research Highlight ,Healthy Volunteers ,Single Molecule Imaging ,3. Good health ,Mutagenesis ,Stem cell ,030217 neurology & neurosurgery ,Cell Division ,Granulocytes - Abstract
Somatic mutations drive the development of cancer and may contribute to ageing and other diseases1,2. Despite their importance, the difficulty of detecting mutations that are only present in single cells or small clones has limited our knowledge of somatic mutagenesis to a minority of tissues. Here, to overcome these limitations, we developed nanorate sequencing (NanoSeq), a duplex sequencing protocol with error rates of less than five errors per billion base pairs in single DNA molecules from cell populations. This rate is two orders of magnitude lower than typical somatic mutation loads, enabling the study of somatic mutations in any tissue independently of clonality. We used this single-molecule sensitivity to study somatic mutations in non-dividing cells across several tissues, comparing stem cells to differentiated cells and studying mutagenesis in the absence of cell division. Differentiated cells in blood and colon displayed remarkably similar mutation loads and signatures to their corresponding stem cells, despite mature blood cells having undergone considerably more divisions. We then characterized the mutational landscape of post-mitotic neurons and polyclonal smooth muscle, confirming that neurons accumulate somatic mutations at a constant rate throughout life without cell division, with similar rates to mitotically active tissues. Together, our results suggest that mutational processes that are independent of cell division are important contributors to somatic mutagenesis. We anticipate that the ability to reliably detect mutations in single DNA molecules could transform our understanding of somatic mutagenesis and enable non-invasive studies on large-scale cohorts. NanoSeq is used to detect mutations in single DNA molecules and analyses show that mutational processes that are independent of cell division are important contributors to somatic mutagenesis.
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- 2021
- Full Text
- View/download PDF
11. Patterns of within-host genetic diversity in SARS-CoV-2
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Dominic P. Kwiatkowski, Grant Hall, Theresa Feltwell, Cristina V. Ariani, Stephen D. Bentley, Rachel Nelson, Inigo Martincorena, Iliana Georgana, Fahad A Khokhar, Michael Spencer Chapman, Andrew R. J. Lawson, Sónia Gonçalves, Gerry Tonkin-Hill, Ewan Harrison, M. Estée Török, Laura G Caller, Luke W. Meredith, Stefanie V Lensing, Surendra Parmar, Naomi R Park, William L Hamilton, Alex Alderton, Moritz Gerstung, David K. Jackson, Myra Hosmillo, Anna Yakovleva, Michael A. Quail, Sarah L Caddy, Charlotte J. Houldcroft, Yasmin Chaudhry, Ian Johnston, Jeffrey C. Barrett, Malte L Pinckert, Aminu S Jahun, Roberto Amato, Ian Goodfellow, John Sillitoe, Martin D. Curran, and Cordelia Langford
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Negative selection ,Mutation rate ,Genetic diversity ,Lineage (genetic) ,Phylogenetic tree ,Evolutionary biology ,Nonsense mutation ,Biology ,Allele frequency ,Genome - Abstract
Monitoring the spread of SARS-CoV-2 and reconstructing transmission chains has become a major public health focus for many governments around the world. The modest mutation rate and rapid transmission of SARS-CoV-2 prevents the reconstruction of transmission chains from consensus genome sequences, but within-host genetic diversity could theoretically help identify close contacts. Here we describe the patterns of within-host diversity in 1,181 SARS-CoV-2 samples sequenced to high depth in duplicate. 95% of samples show within-host mutations at detectable allele frequencies. Analyses of the mutational spectra revealed strong strand asymmetries suggestive of damage or RNA editing of the plus strand, rather than replication errors, dominating the accumulation of mutations during the SARS-CoV-2 pandemic. Within and between host diversity show strong purifying selection, particularly against nonsense mutations. Recurrent within-host mutations, many of which coincide with known phylogenetic homoplasies, display a spectrum and patterns of purifying selection more suggestive of mutational hotspots than recombination or convergent evolution. While allele frequencies suggest that most samples result from infection by a single lineage, we identify multiple putative examples of co-infection. Integrating these results into an epidemiological inference framework, we find that while sharing of within-host variants between samples could help the reconstruction of transmission chains, mutational hotspots and rare cases of superinfection can confound these analyses.
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- 2020
12. Elevated somatic mutation burdens in normal human cells due to defective DNA polymerases
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Sigurgeir Olafsson, Mathijs A. Sanders, Claudia M.A. Pinna, Michael R. Stratton, Philip S. Robinson, Andrew R. J. Lawson, Tim H. H. Coorens, Federico Abascal, Inigo Martincorena, Peter J. Campbell, Henry Lee-Six, Luiza Moore, Ian Tomlinson, Bernard C H Lee, Emily Mitchell, Sara Galvotti, Lynn Martin, Claire Palles, and James Hewinson
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Genetics ,Exonuclease ,Mutation ,Germline mutation ,POLD1 ,biology ,Somatic cell ,DNA polymerase ,biology.protein ,medicine ,Mutation Accumulation ,medicine.disease_cause ,Germline - Abstract
Mutation accumulation over time in normal somatic cells contributes to cancer development and is proposed as a cause of ageing. DNA polymerases Pol ε and Pol δ replicate DNA with high fidelity during normal cell divisions. However, in some cancers defective proofreading due to acquired mutations in the exonuclease domains of POLE or POLD1 causes markedly elevated somatic mutation burdens with distinctive mutational signatures. POLE and POLD1 exonuclease domain mutations also cause familial cancer predisposition when inherited through the germline. Here, we sequenced normal tissue DNA from individuals with germline POLE or POLD1 exonuclease domain mutations. Increased mutation burdens with characteristic mutational signatures were found to varying extents in all normal adult somatic cell types examined, during early embryogenesis and in sperm. Mutation burdens were further markedly elevated in neoplasms from these individuals. Thus human physiology is able to tolerate ubiquitously elevated mutation burdens. Indeed, with the exception of early onset cancer, individuals with germline POLE and POLD1 exonuclease domain mutations are not reported to show abnormal phenotypic features, including those of premature ageing. The results, therefore, do not support a simple model in which all features of ageing are attributable to widespread cell malfunction directly resulting from somatic mutation burdens accrued during life.
- Published
- 2020
13. Osimertinib benefit inEGFR-mutant NSCLC patients withT790M-mutation detected by circulating tumour DNA
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Sarah Smalley, Caroline Caramella, A. Gazzah, Chloe Pannet, Ludovic Lacroix, Nitzan Rosenfeld, M.V. Bluthgen, Vincent Plagnol, Claudio Nicotra, Jordi Remon, Benjamin Besse, Andrew R. J. Lawson, J-C. Soria, Cécile Jovelet, David Planchard, Karen Howarth, Emma Green, Edouard Auclin, and Davina Gale
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Adult ,Male ,0301 basic medicine ,Oncology ,medicine.medical_specialty ,Pathology ,Lung Neoplasms ,DNA Mutational Analysis ,Antineoplastic Agents ,Disease-Free Survival ,Piperazines ,03 medical and health sciences ,T790M ,0302 clinical medicine ,Median follow-up ,Carcinoma, Non-Small-Cell Lung ,Internal medicine ,Biopsy ,Humans ,Medicine ,Osimertinib ,Progression-free survival ,Liquid biopsy ,Lung cancer ,Aged ,Aged, 80 and over ,Acrylamides ,Aniline Compounds ,medicine.diagnostic_test ,business.industry ,DNA, Neoplasm ,Hematology ,Middle Aged ,medicine.disease ,ErbB Receptors ,030104 developmental biology ,Response Evaluation Criteria in Solid Tumors ,030220 oncology & carcinogenesis ,Mutation ,Female ,business - Abstract
Background Approximately 50% of epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer (NSCLC) patients treated with EGFR tyrosine kinase inhibitors (TKIs) will acquire resistance by theT790M mutation. Osimertinib is the standard of care in this situation. The present study assesses the efficacy of osimertinib whenT790M status is determined in circulating cell-free tumour DNA (ctDNA) from blood samples in progressing advancedEGFR-mutant NSCLC patients. Material and methods ctDNAT790M mutational status was assessed by Inivata InVision™ (eTAm-Seq™) assay in 48EGFR-mutant advanced NSCLC patients with acquired resistance to EGFR TKIs without a tissue biopsy between April 2015 and April 2016. ProgressingT790M-positive NSCLC patients received osimertinib (80 mg daily). The objectives were to assess the response rate to osimertinib according to Response Evaluation Criteria in Solid Tumours (RECIST) 1.1, the progression-free survival (PFS) on osimertinib, and the percentage ofT790M positive inctDNA. Results ThectDNAT790M mutation was detected in 50% of NSCLC patients. Among assessable patients, osimertinib gave a partial response rate of 62.5% and a stable disease rate of 37.5%. All responses were confirmed responses. After median follow up of 8 months, median PFS by RECIST criteria was not achieved (95% CI: 4–NA), with 6- and 12-months PFS of 66.7% and 52%, respectively. Conclusion(s) ctDNA from liquid biopsy can be used as a surrogate marker forT790M in tumour tissue.
- Published
- 2017
14. Reliable detection of somatic mutations in solid tissues by laser-capture microdissection and low-input DNA sequencing
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Peter, Ellis, Luiza, Moore, Mathijs A, Sanders, Timothy M, Butler, Simon F, Brunner, Henry, Lee-Six, Robert, Osborne, Ben, Farr, Tim H H, Coorens, Andrew R J, Lawson, Alex, Cagan, Mike R, Stratton, Inigo, Martincorena, and Peter J, Campbell
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Whole Genome Sequencing ,Gene Expression Profiling ,Mutation ,High-Throughput Nucleotide Sequencing ,Humans ,DNA ,Laser Capture Microdissection ,Sequence Analysis, DNA ,Gene Library ,Workflow - Abstract
Somatic mutations accumulate in healthy tissues as we age, giving rise to cancer and potentially contributing to ageing. To study somatic mutations in non-neoplastic tissues, we developed a series of protocols to sequence the genomes of small populations of cells isolated from histological sections. Here, we describe a complete workflow that combines laser-capture microdissection (LCM) with low-input genome sequencing, while circumventing the use of whole-genome amplification (WGA). The protocol is subdivided broadly into four steps: tissue processing, LCM, low-input library generation and mutation calling and filtering. The tissue processing and LCM steps are provided as general guidelines that might require tailoring based on the specific requirements of the study at hand. Our protocol for low-input library generation uses enzymatic rather than acoustic fragmentation to generate WGA-free whole-genome libraries. Finally, the mutation calling and filtering strategy has been adapted from previously published protocols to account for artifacts introduced via library creation. To date, we have used this workflow to perform targeted and whole-genome sequencing of small populations of cells (typically 100-1,000 cells) in thousands of microbiopsies from a wide range of human tissues. The low-input DNA protocol is designed to be compatible with liquid handling platforms and make use of equipment and expertise standard to any core sequencing facility. However, obtaining low-input DNA material via LCM requires specialized equipment and expertise. The entire protocol from tissue reception through whole-genome library generation can be accomplished in as little as 1 week, although 2-3 weeks would be a more typical turnaround time.
- Published
- 2019
15. Somatic mutant clones colonize the human esophagus with age
- Author
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Peter J. Campbell, Kourosh Saeb-Parsy, Federico Abascal, Penny A. Handford, Michael W. J. Hall, Krishnaa T. Mahbubani, Rebecca C. Fitzgerald, Joanna C. Fowler, Agnieszka Wabik, Inigo Martincorena, Michael R. Stratton, Alex Cagan, Kasumi Murai, Andrew R. J. Lawson, Philip H. Jones, Martincorena, Iñigo [0000-0003-1122-4416], Fowler, Joanna C [0000-0001-7546-369X], Wabik, Agnieszka [0000-0001-7231-2343], Lawson, Andrew RJ [0000-0003-3592-1005], Abascal, Federico [0000-0002-6201-1587], Hall, Michael WJ [0000-0003-2904-6902], Mahbubani, Krishnaa [0000-0002-1327-2334], Stratton, Michael R [0000-0001-6035-153X], Handford, Penny A [0000-0002-0590-7651], Saeb-Parsy, Kourosh [0000-0002-0633-3696], Jones, Philip H [0000-0002-5904-795X], and Apollo - University of Cambridge Repository
- Subjects
0301 basic medicine ,Adult ,Male ,Aging ,Esophageal Neoplasms ,Somatic cell ,Mutant ,Biology ,medicine.disease_cause ,DNA sequencing ,Article ,03 medical and health sciences ,Barrett Esophagus ,Young Adult ,Esophagus ,medicine ,Humans ,Receptor, Notch1 ,Selection, Genetic ,Receptor ,Aged ,Mutation ,Multidisciplinary ,Cancer ,Middle Aged ,medicine.disease ,Molecular biology ,Epithelium ,3. Good health ,Clone Cells ,030104 developmental biology ,medicine.anatomical_structure ,Female ,Tumor Suppressor Protein p53 - Abstract
The mutational burden of aging As people age, they accumulate somatic mutations in healthy cells. About 25% of cells in normal, sun-exposed skin harbor cancer driver mutations. What about tissues not exposed to powerful mutagens like ultraviolet light? Martincorena et al. performed targeted gene sequencing of normal esophageal epithelium from nine human donors of varying age (see the Perspective by Chanock). The mutation rate was lower in esophagus than in skin, but there was a strong positive selection of clones carrying mutations in 14 cancer-associated genes. By middle age, more than half of the esophageal epithelium was colonized by mutant clones. Interestingly, mutations in the cancer driver gene NOTCH1 were more common in normal esophageal epithelium than in esophageal cancer. Science , this issue p. 911 ; see also p. 893
- Published
- 2018
16. P3.02b-102 Osimertinib Benefit in ctDNA T790M Positive, EGFR-Mutant NSCLC Patients
- Author
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Vincent Plagnol, Karen Howarth, Emma Green, Chloe Pannet, Nitzan Rosenfeld, Claudio Nicotra, Ludovic Lacroix, David Planchard, Benjamin Besse, Sarah Smalley, Jean-Charles Soria, Caroline Caramella, Andrew R. J. Lawson, Cecile Joelet, A. Gazzah, Maria Bluthgen, Jordi Remon, and Davina Gale
- Subjects
0301 basic medicine ,Pulmonary and Respiratory Medicine ,business.industry ,Mutant ,03 medical and health sciences ,T790M ,030104 developmental biology ,0302 clinical medicine ,Oncology ,030220 oncology & carcinogenesis ,Cancer research ,Medicine ,Osimertinib ,business - Published
- 2017
17. P2.03b-093 Validation and Performance of a Standardized ctDNA NGS Assay across Two Laboratories
- Author
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James H. Clark, Bradley Durham, Nitzan Rosenfeld, Sarah Smalley, Sam Woodhouse, Karen Howarth, Emma Green, Davina Gale, Greg Jones, Shannon Blais, Mikidachi Madi, John Calaway, Michelle Pugh, Tim Forshew, Vincent Plagnol, Esther Musgrave-Brown, and Andrew R. J. Lawson
- Subjects
Pulmonary and Respiratory Medicine ,Oncology ,business.industry ,Medicine ,Computational biology ,business - Published
- 2017
18. RAF gene fusion breakpoints in pediatric brain tumors are characterized by significant enrichment of sequence microhomology
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David W. Ellison, Denise Sheer, Geoffrey Neale, Guy F.L. Hindley, Gabriel A. Jamie, Jing Ma, Ruth G. Tatevossian, Tim Forshew, Gavin P. Kelly, Tania A. Jones, and Andrew R. J. Lawson
- Subjects
DNA Replication ,Male ,Adolescent ,Molecular Sequence Data ,Chromosome Breakpoints ,Sequence alignment ,Minisatellite Repeats ,Astrocytoma ,Biology ,Conserved sequence ,Fusion gene ,Young Adult ,Gene Order ,Genetics ,Humans ,Child ,Gene ,Genetics (clinical) ,Gene Rearrangement ,Base Sequence ,Models, Genetic ,Brain Neoplasms ,Research ,Infant ,Gene rearrangement ,Child, Preschool ,raf Kinases ,Tandem exon duplication ,Gene Fusion ,Homologous recombination ,Sequence Alignment - Abstract
Gene fusions involving members of the RAF family of protein kinases have recently been identified as characteristic aberrations of low-grade astrocytomas, the most common tumors of the central nervous system in children. While it has been shown that these fusions cause constitutive activation of the ERK/MAPK pathway, very little is known about their formation. Here, we present a detailed analysis of RAF gene fusion breakpoints from a well-characterized cohort of 43 low-grade astrocytomas. Our findings show that the rearrangements that generate these RAF gene fusions may be simple or complex and that both inserted nucleotides and microhomology are common at the DNA breakpoints. Furthermore, we identify novel enrichment of microhomologous sequences in the regions immediately flanking the breakpoints. We thus provide evidence that the tandem duplications responsible for these fusions are generated by microhomology-mediated break-induced replication (MMBIR). Although MMBIR has previously been implicated in the pathogenesis of other diseases and the evolution of eukaryotic genomes, we demonstrate here that the proposed details of MMBIR are consistent with a recurrent rearrangement in cancer. Our analysis of repetitive elements, Z-DNA and sequence motifs in the fusion partners identified significant enrichment of the human minisatellite conserved sequence/χ-like element at one side of the breakpoint. Therefore, in addition to furthering our understanding of low-grade astrocytomas, this study provides insights into the molecular mechanistic details of MMBIR and the sequence of events that occur in the formation of genomic rearrangements.
- Published
- 2011
19. Development of a highly sensitive liquid biopsy platform to detect clinically-relevant cancer mutations at low allele fractions in cell-free DNA
- Author
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Karin Schmitt, Sarah Smalley, Karen Howarth, Emma Green, Mikidache Madi, John Calaway, Bradley Durham, Carole A. Foy, Samuel Woodhouse, James H. Clark, Ana Fernandez-Gonzalez, Peter N. Dimitrov, Vincent Plagnol, Greg Jones, Davina Gale, Shannon Blais, Jim F. Huggett, Gerwyn M. Jones, Alexandra S. Whale, Michelle Pugh, Alison S. Devonshire, Michael Epstein, Tim Forshew, Nitzan Rosenfeld, Hadas Raveh-Amit, Andrew R. J. Lawson, Gale, Davina [0000-0002-4521-8199], and Apollo - University of Cambridge Repository
- Subjects
Male ,0301 basic medicine ,Physiology ,Biopsy ,Mutagenesis and Gene Deletion Techniques ,DNA Mutational Analysis ,Gene Identification and Analysis ,lcsh:Medicine ,Artificial Gene Amplification and Extension ,medicine.disease_cause ,Polymerase Chain Reaction ,Biochemistry ,Circulating Tumor DNA ,law.invention ,chemistry.chemical_compound ,Sequencing techniques ,0302 clinical medicine ,law ,Neoplasms ,Medicine and Health Sciences ,Digital polymerase chain reaction ,DNA sequencing ,lcsh:Science ,Polymerase chain reaction ,Mutation ,Multidisciplinary ,High-Throughput Nucleotide Sequencing ,DNA, Neoplasm ,Genomics ,Amplicon ,Neoplastic Cells, Circulating ,Body Fluids ,Blood ,Oncology ,030220 oncology & carcinogenesis ,Female ,Anatomy ,Cell-Free Nucleic Acids ,Transcriptome Analysis ,Research Article ,Adult ,Next-Generation Sequencing ,Surgical and Invasive Medical Procedures ,Computational biology ,Biology ,Research and Analysis Methods ,Sensitivity and Specificity ,Blood Plasma ,03 medical and health sciences ,Cancer detection and diagnosis ,Biomarkers, Tumor ,Genetics ,medicine ,Humans ,Liquid biopsy ,Molecular Biology Techniques ,Mutation Detection ,Molecular Biology ,Gene ,Alleles ,lcsh:R ,Liquid Biopsy ,Reproducibility of Results ,Biology and Life Sciences ,Computational Biology ,Genome Analysis ,Diagnostic medicine ,Mutational Analysis ,030104 developmental biology ,chemistry ,lcsh:Q ,Biomarkers ,DNA - Abstract
Introduction: Detection and monitoring of circulating tumor DNA (ctDNA) is rapidly becoming a diagnostic, prognostic and predictive tool in cancer patient care. A growing number of gene targets have been identified as diagnostic or actionable, requiring the development of reliable technology that provides analysis of multiple genes in parallel. We have developed the InVision™ liquid biopsy platform which utilizes enhanced TAm-Seq™ (eTAm-Seq™) technology, an amplicon-based next generation sequencing method for the identification of clinically-relevant somatic alterations at low frequency in ctDNA across a panel of 35 cancer-related genes. Materials and methods: We present analytical validation of the eTAm-Seq technology across two laboratories to determine the reproducibility of mutation identification. We assess the quantitative performance of eTAm-Seq technology for analysis of single nucleotide variants in clinically-relevant genes as compared to digital PCR (dPCR), using both established DNA standards and novel full-process control material. Results: The assay detected mutant alleles down to 0.02% AF, with high per-base specificity of 99.9997%. Across two laboratories, analysis of samples with optimal amount of DNA detected 94% mutations at 0.25%-0.33% allele fraction (AF), with 90% of mutations detected for samples with lower amounts of input DNA. Conclusions: These studies demonstrate that eTAm-Seq technology is a robust and reproducible technology for the identification and quantification of somatic mutations in circulating tumor DNA, and support its use in clinical applications for precision medicine.
- Published
- 2018
20. Abstract 2743: Comparison of enhanced Tagged-Amplicon Sequencing and digital PCR for circulating tumor DNA analysis in advanced breast cancer
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Matthew Beanney, Sarah Hrebien, Andrew R. J. Lawson, Nicholas C. Turner, Nitzan Rosenfeld, Isaac Garcia-Murillas, Michael Epstein, Karen Howarth, and Emma Green
- Subjects
0301 basic medicine ,COLD-PCR ,Genetics ,Cancer Research ,business.industry ,Advanced breast ,medicine.disease ,03 medical and health sciences ,Exon ,030104 developmental biology ,Breast cancer ,Oncology ,Cancer research ,Medicine ,Coding region ,Digital polymerase chain reaction ,Copy-number variation ,business ,Allele frequency - Abstract
Background Circulating tumor DNA (ctDNA) analysis allows non-invasive detection of tumor mutations and amplifications in advanced breast cancer. Multiple technologies have been developed to analyse ctDNA and here we compared two leading ctDNA detection technologies, enhanced Tagged-Amplicon Sequencing™ (eTAm-Seq™) and digital PCR (dPCR) assays, in advanced breast cancer. Methods We recruited a cohort of 35 women with advanced breast cancer, of whom 23 had two separate blood samples taken in a standard EDTA tube processed immediately or in preservative Streck tubes processed up to 120 hours after venipuncture. Digital PCR was conducted with assays for hotspot actionable mutations in 3 known drivers in breast cancer: PIK3CA exon 9 and 20, ESR1 ligand binding domain and AKT1 (c.49G>A; p.E17K), and ctDNA sequencing was conducted with eTAm-Seq method using a 35-gene panel including cancer hotspots, entire coding regions and copy number variants (CNVs). Results Across both assays, 37 mutations were detected in 35 patients, with PIK3CA mutation in 13 patients (37%), ESR1 mutations in 10 patients (29%), and no AKT1 mutations. ESR1 mutations were polyclonal in 8 patients, with ctDNA eTAm-Seq method revealing substantially more diversity in mutations, with up to 8 individual mutations detected in a patient. There was 96.15% agreement for PIK3CA mutation detection between assays (Kappa 0.89, 95% CI 0.743 to 1.000), 100% agreement for ESR1 mutations (Kappa 1.00, 95% CI 1.000 to 1.000). There was very high correlation in mutation allele frequency between eTAm-Seq and dPCR (r=0.93, 95%CI 0.86 to 0.96, p Conclusions This study demonstrates that ctDNA analysis using eTAmSeq and dPCR have very high agreement in mutation detection in patients with advanced breast cancer patients. Streck tubes present a robust alternative to immediate processing of samples. eTAm-Seq and digital PCR have high clinical validity in mutation detection. Citation Format: Isaac Garcia-Murillas, Matthew Beanney, Michael Epstein, Karen Howarth, Andrew Lawson, Sarah Hrebien, Emma Green, Nitzan Rosenfeld, Nick Turner. Comparison of enhanced Tagged-Amplicon Sequencing and digital PCR for circulating tumor DNA analysis in advanced breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2743. doi:10.1158/1538-7445.AM2017-2743
- Published
- 2017
21. High throughput synthetic lethality screen reveals a tumorigenic role of adenylate cyclase in fumarate hydratase-deficient cancer cells
- Author
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Andrew R. J. Lawson, Jonas Wolf, Tomer Shlomi, Johannes Fredebohm, Michael Boettcher, Jörg D. Hoheisel, Christian Frezza, and Viola Ladenburger
- Subjects
Adenylate kinase ,Synthetic lethality ,Biology ,HLRCC ,Fumarate Hydratase ,03 medical and health sciences ,0302 clinical medicine ,High-throughput RNAi screen ,RNA interference ,Cell Line, Tumor ,Neoplasms ,Cyclic AMP ,Genetics ,Humans ,Adenylate cyclase ,Lethal allele ,RNA, Small Interfering ,Gene Library ,030304 developmental biology ,0303 health sciences ,HEK 293 cells ,Reproducibility of Results ,Molecular biology ,High-Throughput Screening Assays ,3. Good health ,Cell Transformation, Neoplastic ,HEK293 Cells ,Fumarate hydratase-deficiency ,Cell culture ,030220 oncology & carcinogenesis ,Fumarase ,Cancer cell ,Cancer research ,Genes, Lethal ,RNA Interference ,Adenylyl Cyclases ,Research Article ,Biotechnology - Abstract
Background: Synthetic lethality is an appealing technique for selectively targeting cancer cells which have acquired molecular changes that distinguish them from normal cells. High-throughput RNAi-based screens have been successfully used to identify synthetic lethal pathways with well-characterized tumor suppressors and oncogenes. The recent identification of metabolic tumor suppressors suggests that the concept of synthetic lethality can be applied to selectively target cancer metabolism as well. Results: Here, we perform a high-throughput RNAi screen to identify synthetic lethal genes with fumarate hydratase (FH), a metabolic tumor suppressor whose loss-of-function has been associated with hereditary leiomyomatosis and renal cell carcinoma (HLRCC). Our unbiased screen identified synthetic lethality between FH and several genes in heme metabolism, in accordance with recent findings. Furthermore, we identified an enrichment of synthetic lethality with adenylate cyclases. The effects were validated in an embryonic kidney cell line (HEK293T) and in HLRCC-patient derived cells (UOK262) via both genetic and pharmacological inhibition. The reliance on adenylate cyclases in FH-deficient cells is consistent with increased cyclic-AMP levels, which may act to regulate cellular energy metabolism. Conclusions: The identified synthetic lethality of FH with adenylate cyclases suggests a new potential target for treating HLRCC patients.
- Published
- 2014
22. RAF gene fusions are specific to pilocytic astrocytoma in a broad paediatric brain tumour cohort
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Antony Michalski, Ruth G. Tatevossian, Kim Phipps, Tim Forshew, Simon R. Picker, Thomas S. Jacques, Andrew R. J. Lawson, and Denise Sheer
- Subjects
Male ,Proto-Oncogene Proteins B-raf ,Pathology ,medicine.medical_specialty ,Adolescent ,Oncogene Proteins, Fusion ,Astrocytoma ,Biology ,medicine.disease_cause ,Pediatrics ,Pathology and Forensic Medicine ,Cellular and Molecular Neuroscience ,Exon ,Diffuse Astrocytoma ,Glioma ,Gene duplication ,medicine ,Humans ,Neurofibromatosis ,Child ,Pilocytic astrocytoma ,Brain Neoplasms ,Infant ,medicine.disease ,Child, Preschool ,Cancer research ,Female ,Neurology (clinical) ,KRAS - Abstract
Brain tumours are the most common solid tumour in children and are the primary cause of cancer-related death in children and young adults [4, 6]. The most prevalent childhood brain tumours are low-grade gliomas, specifically pilocytic astrocytomas (PAs, WHO Grade I) [1]. PAs are slow-growing tumours which are often cystic, and may occur sporadically or in association with the genetic disorder Neurofibromatosis type 1. Several recent studies including our own have identified novel KIAA1549–BRAF and SRGAP3–RAF1 gene fusions in the majority of PAs tested [3, 7, 8, 12]. In these fusions, the N-terminal autoinhibitory domains of the RAF proteins are replaced by those of KIAA1549 or SRGAP3, resulting in constitutive activation of the ERK/MAPK pathway. A recent study has suggested that the KIAA1549–BRAF fusion is more common in PAs originating in the cerebellum [5]. In low-grade glioma without RAF gene fusions there is increasing evidence for activation of the ERK/MAPK pathway through alternative mechanisms, such as point mutation of KRAS or BRAF [2, 11, 13]. Despite the high frequency of RAF gene fusions in PAs, they have not been investigated in other types of paediatric brain tumours. In this study, we screened a new cohort of 74 paediatric brain tumours, with a range of different pathologies, for all known KIAA1549–BRAF and SRGAP3–RAF1 fusion variants. Access to tumours and clinical data was in accordance with Local Research Ethics Committee (LREC) regulations: Great Ormond Street Hospital LREC reference number 05/Q0508/153. Tumours were classified by diagnostic criteria defined by the World Health Organization (WHO) [10]. Total RNA was extracted from fresh frozen tissue samples using the miRNeasy mini kit (Qiagen, Crawley, UK) and reverse transcribed using the SuperScript First-Strand cDNA synthesis system (Invitrogen, Carlsbad, CA). KIAA1549–BRAF fusions were detected using previously described primers and techniques [3]. The primers used for detecting SRGAP3–RAF1 fusions were 50-TGG CAGTAACCTCATCACCA-30 (located in SRGAP3 exon 10) and 50-GGTTGGGTCGACAACCTTTA-30 (located in RAF1 exon 11). All fusions identified by PCR were confirmed by direct sequencing on a 3100 Genetic Analyzer capillary sequencer (Applied Biosystems, Foster City, CA). Electronic supplementary material The online version of this article (doi:10.1007/s00401-010-0693-y) contains supplementary material, which is available to authorized users.
- Published
- 2010
23. Abstract 3639: Analytical performance and validation of an enhanced TAm-Seq circulating tumor DNA sequencing assay
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Michelle Pugh, Nitzan Rosenfeld, Mikidache Madi, James H. Clark, Vasudev Kumanduri, Sarah Smalley, Davina Gale, Andrew R. J. Lawson, Karen Howarth, Emma Green, Bradley Durham, Vincent Plagnol, Kitty Lo, and Tim Forshew
- Subjects
0301 basic medicine ,Cancer Research ,Cancer ,Biology ,medicine.disease_cause ,medicine.disease ,Molecular biology ,Deep sequencing ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,0302 clinical medicine ,Oncology ,chemistry ,030220 oncology & carcinogenesis ,medicine ,Coding region ,False positive rate ,KRAS ,Allele ,Gene ,DNA - Abstract
Circulating tumor DNA (ctDNA) is becoming established as a tool to supplement conventional biopsies for molecular characterization and monitoring of solid cancers, especially for cancers where tumor tissue is difficult to obtain or is only available at limiting quantity. This requires reliable identification, in patient plasma, of tumor-specific DNA alterations that in some cases may be present as a small fraction of the total cell-free DNA molecules. To overcome these technical challenges, we have developed an enhanced platform for tagged-amplicon deep sequencing (TAm-Seq™). Using a combination of efficient library preparation and statistically-based analysis algorithms, this platform can be used to sequence, identify and quantify cancer mutations across a gene panel including both cancer hotspots, as well as entire coding regions of selected genes. This poster will present validated performance specifications of this multi-gene ctDNA sequencing assay. To perform analytical validation, we used reference standards and plasma DNA controls to demonstrate the sensitivity, specificity and quantitative accuracy of this ctDNA analysis platform. We found that our workflow, using 4 mL input plasma, yields very high sensitivity for variants that are present at allele fraction 0.25% or higher in plasma, and retains substantial sensitivity at allele fractions as low as 0.1%. Using dilution mixtures of well-characterised reference samples, we show that the assay accurately quantifies allele fractions with precision predominantly limited by stochastic sampling. Analysis of plasma samples from control individuals demonstrates a low false positive rate. The assay also detects DNA amplifications (including in ERBB2, MYC, KRAS, EGFR, MET, FGFR1, FGFR2) when the ctDNA are sufficiently high. Together, these data demonstrate the analytical validity and robustness of the TAm-Seq assay and support its use as a basis for clinical applications. Citation Format: Davina Gale, Vincent Plagnol, Andrew Lawson, Michelle Pugh, Sarah Smalley, Karen Howarth, Mikidache Madi, Bradley Durham, Vasudev Kumanduri, Kitty Lo, James Clark, Emma Green, Nitzan Rosenfeld, Tim Forshew. Analytical performance and validation of an enhanced TAm-Seq circulating tumor DNA sequencing assay. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3639.
- Published
- 2016
24. Abstract 3192: Liquid biopsies for molecular profiling of mutations in non-small cell lung cancer patients lacking tissue samples
- Author
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Cécile Jovelet, David Planchard, Chloe Pannet, Emma Green, Nitzan Rosenfeld, Ludovic Lacroix, Ken Oulassen, Andrew R. J. Lawson, Sarah Smalley, Vincent Plagnol, Nathalie Chaput, Jordi Remon, Benjamin Besse, David Gale, Kenth Howarth, A. Gazzah, and Jean-Charles Soria
- Subjects
0301 basic medicine ,Cancer Research ,Pathology ,medicine.medical_specialty ,Crizotinib ,medicine.diagnostic_test ,business.industry ,medicine.disease ,medicine.disease_cause ,03 medical and health sciences ,T790M ,030104 developmental biology ,Oncology ,Biopsy ,medicine ,Cancer research ,Adenocarcinoma of the lung ,Adenocarcinoma ,KRAS ,Liquid biopsy ,business ,Lung cancer ,medicine.drug - Abstract
Introduction: Approximately 30% of patients with an adenocarcinoma of the lung have an actionable driver mutation. Further understanding the molecular mechanisms of acquired resistance to targeted therapies provides key information for determining subsequent treatment options. Access to tumor tissue to perform either the initial molecular profile or at the point of acquired resistance, however, is often limited. Circulating tumor DNA (ctDNA) can be used as a minimally invasive method for the detection and quantification of molecular abnormalities. We performed a prospective study to assess molecular alterations in the ctDNA of NSCLC patients in whom the initial molecular profile or profile at acquired resistance was unknown due to lack of tumor tissue biopsy or insufficient cellularity in the biopsy. Methods: Plasma samples were collected from 52 pre-treated advanced NSCLC patients at the Gustave Roussy. DNA was extracted from < 5 ml of plasma and analysed using Inivata's enhanced TAm-SeqTM assay covering regions from 35 cancer-related genes. Sequences were generated using Illumina sequencing. We also analysed plasma taken following treatments prescribed after the original molecular profile detected using plasma ctDNA. Results: From July 2015 to October 2015, 52 patients were included (63% female, 37% never-smoker, 95% diagnosed with an adenocarcinoma subtype, 95% with stage IV disease, and 54% had EGFR mutant tumors of which 68% had mutations in exon 19 and 32% had mutations in exon 21). ctDNA profiling was successfully performed for all patients, and mutations were detected in 38 of 52 patients. The median number of mutations detected in plasma samples was 1. Within the EGFR mutant subpopulation, T790M mutations were identified including 8 acquired cases (with a concomitant C797S mutation in 1 case) and 1 primary T790M mutation. Of these patients, 5 started personalised treatment with AZD9291 based on the results of ctDNA analysis. In the other 18 patients with EGFR mutant tumors, no acquired mutations associated with resistance were detected. Other results encompassed: 2 plasma samples with EGFR mutation exon 18 (G719A, G719C) leading to initiation of afatinib in one case, 1 case with EGFR mutation exon 21 (L861Q), 1 patients with ERBB2 exon 20 insertion, 3 KRAS mutant detected in plasma (G12C, G12S, G12F), 2 STK11 mutant samples, and 1 patient with a MET mutation (exon 14) who subsequently started crizotinib. Conclusions: ctDNA analysis with Inivata's enhanced TAm-Seq™ provides an alternative method of ‘liquid biopsy’ for obtaining molecular profile of mutations present in NSCLC patients in the absence of an invasive tissue biopsy. Liquid biopsy identified cancer mutations in 73% of the study population, and 18% of those patients subsequently received treatment tailored to the plasma ctDNA detected mutations. An update on the analysis of 75 patients will be presented during the conference. Citation Format: Jordi Remon, Jean Charles Soria, David Planchard, Cecile Jovelet, Chloe Pannet, Ludovic Lacroix, Annas Gazzah, Andrew Lawson, Sarah Smalley, Kenth Howarth, David Gale, Emma Green, Vincent Plagnol, Nitzan Rosenfeld, Ken Oulassen, Nathalie Chaput, Benjamin Besse. Liquid biopsies for molecular profiling of mutations in non-small cell lung cancer patients lacking tissue samples. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3192.
- Published
- 2016
25. Evaluation of liquid biopsies for molecular profiling and monitoring in non-small cell lung cancer (NSCLC) patients
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Jean-Charles Soria, Cécile Jovelet, Andrew R. J. Lawson, Benjamin Besse, Ludovic Lacroix, Nitzan Rosenfeld, Chloe Pannet, Sarah Smalley, Davina Gale, Jordi Remon, Vincent Plagnol, Karen Howarth, and Emma Green
- Subjects
Oncology ,Cancer Research ,medicine.medical_specialty ,business.industry ,Internal medicine ,medicine ,Profiling (information science) ,non-small cell lung cancer (NSCLC) ,medicine.disease ,business ,respiratory tract diseases - Abstract
11533Background: Effective and non-invasive methods for actionable molecular alterations in NSCLC patients (pts) are urgently needed. Molecular profiling is often limited by access to sufficient tu...
- Published
- 2016
26. Abstract B7: Assessing the clinical applications of ctDNA in patients with advanced stage metastatic cancer using our enhanced TAm-Seq platform
- Author
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Nitzan Rosenfeld, K. Howarth, Tim Forshew, Andrew R. J. Lawson, Davina Gale, Mehdi Touat, Benjamin Besse, Jordi Remon-Masip, Sarah Smalley, Jean-Charles Soria, Esperanza Perez, Vincent Plagnol, Ludovic Lacroix, Michelle Pugh, and Antoine Hollebecque
- Subjects
Oncology ,Cancer Research ,medicine.medical_specialty ,medicine.diagnostic_test ,business.industry ,medicine.medical_treatment ,Cancer ,Bioinformatics ,medicine.disease ,Radiation therapy ,T790M ,medicine.anatomical_structure ,Internal medicine ,Biopsy ,medicine ,Mutation frequency ,Vemurafenib ,business ,Allele frequency ,Lymph node ,medicine.drug - Abstract
We have used Inivata's enhanced tagged-amplicon deep sequencing (TAm-Seq™) platform to harness the power of circulating tumor DNA (ctDNA) to accurately identify low level mutations in multiple cancers, monitor the progression of disease and track patient response to therapy. We have developed an improved TAm-Seq platform, which enables detection of point mutations and indels in ctDNA with high levels of sensitivity and specificity. Our standardised TAm-Seq platform has a rapid turnaround time and is performed on plasma obtained from blood collected in a single 9mL standard EDTA tube. The TAm-Seq assay is able to analyze regions of interest (either hotspots or entire coding regions) in 35 distinct genes in the human genome with such sensitivity as to detect mutations at an allele frequency less than 1% with unpublished data demonstrating sensitivity as low as 0.1%. We performed longitudinal analyses on a series of case studies in different types of cancer to determine molecular profiles and monitor treatment response. Patient A is a stage IV NSCLC patient with bone metastases. The patient was unresponsive to initial treatment, with a suspected T790M EGFR mutation. Patient B is a BRAF V600E-positive cerebral anaplastic xanthoastrocytoma patient with first-line treatment of surgery and radiotherapy, presenting at relapse with rare multiple extra-cranial (lymph node and bone) metastases. An initial cohort of non-small cell lung cancer (NSCLC) patients, including the Patient A case study, had blood drawn at 2 time points (Day 1 and Day 21) which were analysed using TAm-Seq demonstrating clinical response correlation to reduction in mutant alleles between pre- and post-treatment. In addition to this, Patient A was confirmed by TAm-Seq as having the suspected T790M EGFR mutation at a 0.57% frequency. As tumor biopsy tissue was unavailable for testing resistant mutations in this instance, the detection of the T790M mutation by TAm-Seq enabled the patient to become eligible to receive AZD9291 off-label by special permission. The patient went on to show a complete metabolic response on PET scan. Patient B, previously presenting with a BRAF V600E mutant xanthoastrocytoma, was initiated off-label vemurafenib with ctDNA analysis detecting mutation frequency at 3.45% prior to treatment and 2.16% following 2 weeks of treatment. Follow-up samples have been collected at multiple time points for patient A and B with testing in progress. Additional case studies are in progress and all data will be presented. This work demonstrates the potential utility of using ctDNA to identify resistance mutations and monitor treatment response of a tumor when a conventional biopsy is unobtainable. It highlights the importance of high sensitivity mutation detection and analyzing multiple genomic regions, as such a high proportion of ctDNA mutations in our data were present at an allele frequency of Citation Format: Sarah K. Smalley, Davina Gale, Andrew RJ Lawson, Jordi Remon-Masip, Esperanza Perez, Michelle Pugh, Karen Howarth, Mehdi Touat, Jean-Charles Soria, Antoine Hollebecque, Benjamin Besse, Tim Forshew, Vincent Plagnol, Ludovic Lacroix, Nitzan Rosenfeld. Assessing the clinical applications of ctDNA in patients with advanced stage metastatic cancer using our enhanced TAm-Seq platform. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B7.
- Published
- 2015
27. MYB upregulation and genetic aberrations in a subset of pediatric low-grade gliomas
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Sheila A. Shurtleff, Amar Gajjar, Ruth G. Tatevossian, Denise Sheer, Simon Bailey, James Dalton, Andrew R. J. Lawson, Geoff Neale, David W. Ellison, Bo Tang, Jing Ma, Tim Forshew, and Suzanne J. Baker
- Subjects
Male ,Adolescent ,Biology ,Oncogene Proteins v-myb ,Article ,Pathology and Forensic Medicine ,Cohort Studies ,Cellular and Molecular Neuroscience ,Age Distribution ,Glioma ,Gene duplication ,medicine ,Humans ,MYB ,Oligodendroglial Tumor ,Genetic Predisposition to Disease ,Child ,neoplasms ,Chromosome Aberrations ,Oncogene ,medicine.diagnostic_test ,Brain Neoplasms ,Age Factors ,Gene Amplification ,medicine.disease ,Molecular biology ,nervous system diseases ,Up-Regulation ,Child, Preschool ,Mutation ,Cancer research ,Immunohistochemistry ,Female ,Neurology (clinical) ,Gene Deletion ,Fluorescence in situ hybridization - Abstract
Recent studies of genetic abnormalities in pediatric low-grade gliomas (LGGs) have focused on activation of the ERK/MAPK pathway by KIAA1549-BRAF gene fusions in the majority of pilocytic astrocytomas (PAs) and by rare mutations in elements of the pathway across histopathologically diverse LGGs. This study reports that MYB, an oncogene not previously implicated in gliomagenesis, is activated in a diverse subset of pediatric LGGs. The study cohort comprised 57 pediatric LGGs and a comparative cohort of 59 pediatric high-grade gliomas (HGGs). The LGG cohort included 34 PAs and 23 diffuse gliomas; fibrillary astrocytomas (n = 14), oligodendroglial tumors (n = 7), and angiocentric gliomas (n = 2). MYB copy number abnormalities were disclosed using Affymetrix 6.0 SNP arrays and confirmed using interphase fluorescence in situ hybridization. Novel MYB amplifications that upregulate MYB RNA and protein expression were demonstrated in 2/14 diffuse astrocytomas. In addition, focal deletion of the terminal region of MYB was seen in 1 of 2 angiocentric gliomas (AGs). Increased expression of MYB was demonstrated by quantitative RT-PCR and immunohistochemistry. MYB upregulation at the protein level was demonstrated in a proportion of diffuse LGGs (60%), pilocytic astrocytomas (41%), and HGGs (19%), but abnormalities at the genomic level were only a feature of diffuse gliomas. Our data suggest that MYB may have a role in a subset of pediatric gliomas, through a variety of mechanisms in addition to MYB amplification and deletion.
- Published
- 2010
28. Activation of the ERK/MAPK pathway: a signature genetic defect in posterior fossa pilocytic astrocytomas
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Rowena L Carter, Babatunji W Ogunkolade, Jing Ma, Tania A. Jones, James Dalton, Bryan D. Young, Geoff Neale, Amar Gajjar, David W. Ellison, Denise Sheer, Ruth G. Tatevossian, Alberto Broniscer, Andrew R. J. Lawson, Tim Forshew, Simon Bailey, Johan Aarum, and Tracy Chaplin
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MAPK/ERK pathway ,Adult ,Proto-Oncogene Proteins B-raf ,DNA, Complementary ,Adolescent ,Oncogene Proteins, Fusion ,MAP Kinase Signaling System ,DNA Mutational Analysis ,Biology ,Astrocytoma ,medicine.disease_cause ,Pathology and Forensic Medicine ,Fusion gene ,Young Adult ,Glioma ,medicine ,Humans ,Kinase activity ,Child ,neoplasms ,Mutation ,Pilocytic astrocytoma ,Brain Neoplasms ,GTPase-Activating Proteins ,Infant ,medicine.disease ,digestive system diseases ,Enzyme Activation ,Proto-Oncogene Proteins c-raf ,Child, Preschool ,Cancer research ,KRAS ,Mitogen-Activated Protein Kinases - Abstract
We report genetic aberrations that activate the ERK/MAP kinase pathway in 100% of posterior fossa pilocytic astrocytomas, with a high frequency of gene fusions between KIAA1549 and BRAF among these tumours. These fusions were identified from analysis of focal copy number gains at 7q34, detected using Affymetrix 250K and 6.0 SNP arrays. PCR and sequencing confirmed the presence of five KIAA1549-BRAF fusion variants, along with a single fusion between SRGAP3 and RAF1. The resulting fusion genes lack the auto-inhibitory domains of BRAF and RAF1, which are replaced in-frame by the beginning of KIAA1549 and SRGAP3, respectively, conferring constitutive kinase activity. An activating mutation of KRAS was identified in the single pilocytic astrocytoma without a BRAF or RAF1 fusion. Further fusions and activating mutations in BRAF were identified in 28% of grade II astrocytomas, highlighting the importance of the ERK/MAP kinase pathway in the development of paediatric low-grade gliomas.
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- 2009
29. MAPK pathway activation and the origins of pediatric low-grade astrocytomas
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Guy F.L. Hindley, Denise Sheer, Ruth G. Tatevossian, Andrew R. J. Lawson, David W. Ellison, and Tim Forshew
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Proto-Oncogene Proteins B-raf ,MAPK/ERK pathway ,medicine.medical_specialty ,Adolescent ,MAP Kinase Signaling System ,Physiology ,Clinical Biochemistry ,Brain tumor ,Astrocytoma ,Biology ,Gene Expression Regulation, Enzymologic ,Fusion gene ,Molecular genetics ,medicine ,Humans ,Child ,Gene ,Regulation of gene expression ,Brain Neoplasms ,Infant, Newborn ,Infant ,Cell Biology ,medicine.disease ,Enzyme Activation ,Gene Expression Regulation, Neoplastic ,Child, Preschool ,Immunology ,Cancer research ,Gene Fusion ,Mitogen-Activated Protein Kinases - Abstract
Low-grade astrocytomas (LGAs) are the most common type of brain tumor in children. Until recently, very little was known about the underlying biology and molecular genetics of these tumors. However, within the past year a number of studies have shown that the MAPK pathway is constitutively activated in a high proportion of LGAs. Several genetic aberrations which generate this deregulation of the MAPK pathway have been identified, most notably gene fusions between KIAA1549 and BRAF. In this review we summarize these findings, discuss how these gene fusions may arise and consider possible implications for diagnosis and treatment.
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- 2009
30. Abstract 2412: Assessment of clinical applications of circulating tumor DNA using an enhanced TAm-Seq platform
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Andrew R. J. Lawson, Abdelaziz Fahem, Nitzan Rosenfeld, Tim Forshew, Vincent Plagnol, James D. Brenton, and Davina Gale
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Cancer Research ,Oncology ,Circulating tumor DNA ,Computational biology ,Biology ,Molecular biology - Abstract
Novel biomarkers are required to assess tumor burden and response in cancer as conventional biopsies are invasive, costly and only provide a snapshot of the mutational profile at a given time and location. A promising biomarker is the detection of genomic material released from tumors into the blood plasma of patients, known as circulating tumor DNA (ctDNA). ctDNA has been detected in plasma for a wide range of solid tumors and can be distinguished from other (germline) cell-free DNA by the presence of tumor-specific DNA alterations or known hotspot mutations. However, the potential of ctDNA as a biomarker has not yet been fully realized due to technical challenges associated with its detection and analysis, including the short fragment sizes (140-170 bp), small number of amplifiable copies and low/variable allele fractions of ctDNA. We have developed an enhanced platform for tagged-amplicon deep sequencing (TAm-Seq™). Using a combination of improved library preparation and bespoke data analysis methods, this platform can be used to sequence established cancer hotspots and the entire coding regions of selected genes, while preserving high levels of specificity and sensitivity. Using this approach, we have developed an assay that analyzes ∼20 kb of the genome (including regions of interest in more than 30 genes) with sensitivity down to a few mutant copies. Performance of this assay has been demonstrated using spike-in experiments, dilution series and clinical sample cohorts. Proof of concept studies have shown the potential of ctDNA to be used to assess tumor mutation status, monitor tumor dynamics, assess response to treatment and identify mutations associated with acquired drug resistance and disease progression. This non-invasive approach - a “liquid biopsy” - offers a revolution in how cancer can be detected, monitored and treated. Citation Format: Andrew RJ Lawson, Vincent Plagnol, Abdelaziz Fahem, Tim Forshew, James D. Brenton, Davina Gale, Nitzan Rosenfeld. Assessment of clinical applications of circulating tumor DNA using an enhanced TAm-Seq platform. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2412. doi:10.1158/1538-7445.AM2015-2412
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- 2015
31. Assessment of clinical applications of circulating tumor DNA using an enhanced TAm-Seq platform
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Abdelaziz Fahem, Nitzan Rosenfeld, Andrew R. J. Lawson, Vincent Plagnol, Tim Forshew, James D. Brenton, and Davina Gale
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Cancer Research ,Oncology ,Circulating tumor DNA ,business.industry ,Tumor burden ,Snapshot (computer storage) ,Medicine ,Computational biology ,Bioinformatics ,business - Abstract
e22057 Background: Novel biomarkers are required to assess tumor burden and response in cancer as conventional biopsies are invasive, costly and only provide a snapshot of the mutational profile at...
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- 2015
32. Molecular analysis of pediatric brain tumors identifies microRNAs in pilocytic astrocytomas that target the MAPK and NF-κB pathways
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Muhammad A. Mumin, Denise Sheer, Kim Phipps, Sheena N. Patel, Andrew R. J. Lawson, Simon R. Picker, Thomas S. Jacques, Antony Michalski, Gabriel T. Doctor, Jennie N. Jeyapalan, Tania A. Jones, Ruth G. Tatevossian, and Tim Forshew
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MAPK/ERK pathway ,Grade I astrocytoma ,Male ,Pathology ,medicine.medical_specialty ,IGFBP7 ,Adolescent ,Biology ,Astrocytoma ,Pathology and Forensic Medicine ,miR-155 ,Cellular and Molecular Neuroscience ,Neuroinflammation ,CDKN2A ,microRNA ,medicine ,CEBPB ,Humans ,RNA, Messenger ,Child ,Children ,neoplasms ,Oligonucleotide Array Sequence Analysis ,Mitogen-Activated Protein Kinase Kinases ,Brain Neoplasms ,Research ,Gene Expression Profiling ,NF-kappa B ,Infant ,KIAA1549-BRAF ,medicine.disease ,Gene expression profiling ,miR-146a ,MicroRNAs ,nervous system ,Senescence-associated secretory phenotype (SASP) ,Child, Preschool ,Cancer research ,Female ,Neurology (clinical) ,Signal Transduction - Abstract
Introduction Pilocytic astrocytomas are slow-growing tumors that usually occur in the cerebellum or in the midline along the hypothalamic/optic pathways. The most common genetic alterations in pilocytic astrocytomas activate the ERK/MAPK signal transduction pathway, which is a major driver of proliferation but is also believed to induce senescence in these tumors. Here, we have conducted a detailed investigation of microRNA and gene expression, together with pathway analysis, to improve our understanding of the regulatory mechanisms in pilocytic astrocytomas. Results Pilocytic astrocytomas were found to have distinctive microRNA and gene expression profiles compared to normal brain tissue and a selection of other pediatric brain tumors. Several microRNAs found to be up-regulated in pilocytic astrocytomas are predicted to target the ERK/MAPK and NF-κB signaling pathways as well as genes involved in senescence-associated inflammation and cell cycle control. Furthermore, IGFBP7 and CEBPB, which are transcriptional inducers of the senescence-associated secretory phenotype (SASP), were also up-regulated together with the markers of senescence and inflammation, CDKN1A (p21), CDKN2A (p16) and IL1B. Conclusion These findings provide further evidence of a senescent phenotype in pilocytic astrocytomas. In addition, they suggest that the ERK/MAPK pathway, which is considered the major driver of these tumors, is regulated not only by genetic aberrations but also by microRNAs. Electronic supplementary material The online version of this article (doi:10.1186/s40478-015-0266-3) contains supplementary material, which is available to authorized users.
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33. Liquid biopsies could be superior to tumor biopsy to provide a molecular profile in non-small cell lung cancer (NSCLC) patients
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Emma Green, Cécile Jovelet, Sarah Smalley, David Planchard, Jean-Charles Soria, Andrew R. J. Lawson, Chloe Pannet, Vincent Plagnol, David Gale, Nicolas Rosenfeld, Ludovic Lacroix, Benjamin Besse, Kevin Howarth, and Jordi Remon
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FACE EDEMA ,Oncology ,Pulmonary and Respiratory Medicine ,medicine.medical_specialty ,Crizotinib ,business.industry ,non-small cell lung cancer (NSCLC) ,macromolecular substances ,medicine.disease ,Rash ,Gastroenterology ,Decreased appetite ,carbohydrates (lipids) ,stomatognathic diseases ,Internal medicine ,otorhinolaryngologic diseases ,medicine ,Vomiting ,bacteria ,Tumor biopsy ,Molecular Profile ,medicine.symptom ,business ,medicine.drug - Abstract
vomiting (25%), and fatigue (23%). Most AEs were Grade (G) 1-2. The G3 treatment-related AEs were rash (6 pts), fatigue (1 pt), decreased appetite (1 pt), dehydration (1 pt), pruritus (1 pt), and face edema (1 pt). In particular, no G3 treatment-related gastrointestinal toxicity or liver enzyme elevation has been reported. To date, 24 ALKþ NSCLC pts treated at doses 200 mg are evaluable for response; partial response (PR) was achieved in 16 pts (67%) and stable disease (SD) in 3 pts (13%). In the crizotinib-naive pts (n1⁄48), responses were observed in 6 pts (75%) and SD in 1 pt (13%). In the 12 pts with prior crizotinib but no other ALK TKI, 10 pts (83%) achieved PR and 1 (8%) SD. CNS responses have been observed in both crizotinib naive and crizotinib resistant pts. The median duration of treatment in the 24 evaluable ALKþ pts is 23.8þ weeks, with the longest being 112þ weeks. Conclusion: X-396 is well-tolerated and induces responses in both crizotinib-naive and crizotinib-resistant ALKþ NSCLC pts, as well as patients with CNS lesions. Enrollment is ongoing in the expansion cohorts.
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